Antibacterial activities of five cationic gemini surfactants with ethylene glycol bisacetyl spacers

Article abstract of DOI:10.1007/s11743-014-1620-3

A series of cationic gemini surfactants containing two dimethylalkylammonium chains linked by ethylene glycol bisacetyl spacers were synthesized [G _m-AnA-m, G = gemini surfactant, m = 12 (-C₁₂H₂₅), 14 (-C₁₄H₂₉), or 16 (-C₁₆H₃₃), A = acetyl, and n = 2, 3, or 4 is the number of ethylene glycol units in the spacers]. Because of the inductive effect of the oxygen atom in the spacer, acylation can take place using chloroacetyl chloride instead of bromoacetyl bromide which helps to limit the use of environmentally harmful reagents. Critical micelle concentrations were determined using conductivity measurements. The antibacterial activities of the surfactants against Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli were evaluated from the minimum inhibitory concentration (MIC), minimum bacterial concentration, a time-kill study, and the inhibitory zone. Increasing the length of the spacer did not result in an obvious change of antibacterial activity. However, increasing the length of the alkyl chain apparently increased the antibacterial activity against S. aureus but decreased the antibacterial activity against E. coli. The G_12-A2A-12 surfactant had the lowest CMC of 1.26 mmol L^-1 and exhibited the best antibacterial activity with a MIC of 32 μg mL^-1 toward S. aureus and 64 μg mL^-1 toward E. coli in the presence of 10⁵ CFU of bacteria. This work indicated that these cationic gemini surfactants have potential applications as antibacterial agents and emulsifiers.

Full text of DOI:10.1007/s11743-014-1620-3

J Surfact Deterg
DOI 10.1007/s11743-014-1620-3
ORIGINAL ARTICLE
Antibacterial Activities of Five Cationic Gemini Surfactants
with Ethylene Glycol Bisacetyl Spacers
Shengnan Guo
Jinzhi Zhang
•
Xiaowen Sun
Hong Ni
•
Qichao Zou
•
•
Received: 25 November 2013 / Accepted: 14 July 2014
Ó AOCS 2014
-
1
Abstract A series of cationic gemini surfactantscontaining
two dimethylalkylammonium chains linked by ethylene
of 1.26 mmol L
and exhibited the best antibacterial
-1
activity with a MIC of 32 lg mL toward S. aureus and
-1
5
glycol bisacetyl spacers were synthesized [Gm-AnA-m
,
64 lg mL toward E. coli in the presence of 10 CFU of
bacteria. This work indicated that these cationic gemini sur-
factants have potential applications as antibacterial agents
and emulsifiers.
G = gemini surfactant, m = 12 (–C H ), 14 (–C H ), or
1
2
25
14 29
1
6 (–C H ), A = acetyl, and n = 2, 3, or 4 is the number of
16 33
ethylene glycol units in the spacers]. Because of the inductive
effect of the oxygen atom in the spacer, acylation can take
place using chloroacetyl chloride instead of bromoacetyl
bromide which helps to limit the use of environmentally
harmful reagents. Critical micelle concentrations were
determined using conductivity measurements. The antibac-
terial activities of the surfactants against Gram-positive
bacterium Staphylococcus aureus and Gram-negative bac-
terium Escherichia coli were evaluated from the minimum
inhibitory concentration (MIC), minimum bacterial concen-
tration, a time–kill study, and the inhibitory zone. Increasing
the length of the spacer did not result in an obvious change of
antibacterial activity. However, increasing the length of the
alkyl chain apparently increased the antibacterial activity
against S. aureus but decreased the antibacterial activity
against E. coli. The G12-A2A-12 surfactant had thelowestCMC
Keywords Quaternary ammonium salt Á Cationic gemini
surfactant Á Ethylene glycol bisacetyl spacer Á Spacer chain
length Á Alkyl chain length Á Antibacterial activity
Abbreviations
QACs
Quaternary ammonium compounds
G12-A2A-12 Diethylene glycol bisacetateyl-a,x-bis
(dimethydodecylammonium choride)
G12-A3A-12 Triethylene glycol bisacetateyl-a,x-bis
(dimethydodecylammonium choride)
G12-A4A-12 Tetraethylene glycol bisacetateyl-a,x-bis
(dimethydodecylammonium choride)
G14-A2A-14 Diethylene glycol bisacetateyl-a,x-bis
(dimethytetradecylammonium choride)
G16-A2A-16 Diethylene glycol bisacetateyl-a,x-bis
(
dimethyhexadecylammonium choride)
1
1
H NMR
H Nuclear magnetic resonance
S. Guo and X. Sun contributed equally to this work.
FTIR
CMC
acb
Fourier transform infrared spectroscopy
Critical micelle concentration
Counterion dissociation constant
Counterion binding coefficient
Escherichia coli
Electronic supplementary material The online version of this
article (doi:10.1007/s11743-014-1620-3) contains supplementary
material, which is available to authorized users.
bcb
E. coli
S. aureus
MIC
S. Guo Á Q. Zou Á J. Zhang
Staphylococcus aureus
Faculty of Chemistry and Chemical Engineering,
Hubei University, Wuhan 430062, China
Minimum inhibitory concentration
Minimum bacterial concentration
Tryptic soy agar
MBC
TSA
X. Sun Á H. Ni (&)
Faculty of Life Science, Hubei University,
Wuhan 430062, China
e-mail: nh64@sina.com
TSB
Tryptic soy broth
LPS
Lipopolysaccharide
1
23

J Surfact Deterg
Introduction
that the charge densities of these surfactants are higher than
those of conventional surfactants. Therefore, it is reasonable
to assume that gemini surfactants would be able to more
rapidly adsorb to the bacteria surface than conventional sur-
factants and result in bacteria death. There are many reports
discussing the antimicrobial activity of QACs, but a complete
understanding of their structure–activity relationship is lack-
ing [23]. In this work, the effects of different lengths of the
alkyl chain and spacer chain in five gemini surfactants on the
bactericidal activities were evaluated by determining the
minimum inhibitory concentration (MIC) and minimum
bacterial concentration (MBC) of the surfactants, performing
a time–kill study, and evaluating the inhibitory zone against
the Gram-positive bacterium Staphylococcus aureus and the
Gram-negative bacterium Escherichia coli.
We are inevitably exposed to a variety of bacteria, fungi,
and other microorganisms. The resistance of pathogens to
antibacterial agents is a growing concern in public health
[
1]. Antimicrobial materials are a group of functional
materials that possess properties that can kill or inhibit
microbial growth and have applications in medicine,
household articles, and food packaging among others [2,
3
]. However, like other chemical syntheses, methods for
the preparation of antibacterial materials are sought which
minimize the impact on the environment.
During the past two decades, numerous antimicrobial
agents, such as quaternary ammonium compounds (QACs)
[
4, 5], fluoroquinolones [6], oxazolidinones [7], antibacte-
rial polymers [8, 9], and transition metal complexes [10,
1], have been developed. Among these, QACs and poly-
1
Experimental
mers with QA substituents have been extensively used
because of their many merits. In general, QACs possess
both a positively charged hydrophilic ammonium group
and a long hydrophobic alkyl chain [12] which provides a
hydrophobic segment compatible with the bilayer of the
outer cell wall [13, 14]. QACs have several advantages
over other antibacterial agents, including excellent cell
membrane penetration properties, low toxicity, environ-
mental stability, low skin irritability, low corrosiveness,
extended residence time, and favorable biological activity.
The antibacterial activity of QACs strongly depends on the
nature of the organic groups attached to the nitrogen, the
number of nitrogen atoms present, and the alkyl chain
length. Many experiments have shown that QACs con-
taining one long alkyl chain substituent of at least eight
carbon atoms are effective biocides [15]. However, the use
of conventional QACs is still limited by the development
of microbial resistance [16]. Recently, particular attention
has been directed toward cationic gemini surfactants as
new types of QACs which exhibit better antibacterial
activities than conventional surfactants [17, 18].
Materials
Chloroacetyl chloride was purchased from Shanghai Jinshan
Ting New Chemical Plant Reagent Factory, Shanghai,
China. Monochloroacetic acid was purchased from Chengdu
Kelong Chemical Reagent Factory, Chengdu, China.
Diethylene glycol, triethylene glycol, tetraethylene glycol,
dodecyldimethyl tertiary amine, tetradecyldimethyl tertiary
amine, and hexadecyldimethyl tertiary amine were pur-
chased from Aladdin, Shanghai, China. Acetone and petro-
leum ether were obtained from Tianjin Bodi Chemical Co.
Ltd., Tianjin, China. All chemicals were reagent grade and
used without further purification except for acetone which
was purified by reflux distillation. The bacterial strains used
for antibacterial activity tests, S. aureus RN4220 and E. coli
TOP10, were by the Faculty of Life Science, Hubei Uni-
versity, Wuhan, China. Tryptic soy agar (TSA) and tryptic
soy broth (TSB) were purchased from Aladdin, Shanghai,
China. Sodium chloride was purchased from Sinopharm
Chemical Reagent Co., Ltd., Tianjin, China. Peptone and
yeast extract powder were purchased from Beijing Twin
Twist Microbial Culture Medium Products Factory, Beijing,
China. Agar was purchased from Biosharp, Japan. The
Gemini surfactants are bis-QACs composed of two
hydrophilic polar head groups and two hydrophobic hydro-
carbonchainscovalently connectedbya spacer[19, 20]. Their
generally accepted mechanism of action is that the cationic
sites of the QACs are adsorbed onto anionic sites of the cell
wall surface by electrostatic interactions. Then a significant
lipophilic component promotes diffusion through the cell wall
and the QAC binds to the cell membrane, disrupting the
cytoplasmic membrane and causing the release of electrolytes
and nucleic materials which results in bacteria death [21, 22].
Adsorption to negatively charged bacterial cell surfaces is
expected to increase with an increase in the charge density of
the cationic biocides. Because of the spacer chain in gemini
surfactants, the two hydrophilic polar head groups are so close
96-well microtiter plates were from WHB, Shanghai, China.
Synthesis
Synthesis of Diethylene Glycol-bis(2-Chloroethanoate),
Triethylene Glycol-bis(2-Chloroethanoate),
and Tetraethylene Glycol-bis(2-Chloroethanoate)
(Step 1: Acylation)
Monochloroacetic acid (0.6 mol) and chloroacetyl chloride
(0.88 mol) were added to a 250-mL flask equipped with
1
23

J Surfact Deterg
reflux condenser, dropping funnel, and thermometer fixed
on a magnetic stirrer. The solution mixture was stirred and
the temperature was adjusted to 55 °C to dissolve the
monochloroacetic acid completely, and then diethylene,
triethylene, or tetraethylene glycol (0.4 mol) was added
from a dropping funnel over 2 h. After 1 h, the reaction
was stopped and the mixture was poured into ice water
with stirring until the excess chloroacetyl chloride was
completely hydrolyzed. Finally, the ethylene glycol-bis(2-
chloroethanoate) was obtained by extraction, separation,
and drying. The light yellow liquids, diethylene glycol-
bis(2-chloroethanoate) (65.7 %), triethylene glycol-bis(2-
chloroethanoate) (63.2 %), and tetraethylene glycol-bis(2-
chloroethanoate) (57.8 %), were obtained in the indicated
yields [24].
Synthesis of Ethylene Glycol Bisacetyl-a,x-
bis(dimethylalkylammonium Choride) (Step 2:
Quaternization)
Scheme 1 Synthesis of bis-quaternary ammonium cationic gemini
surfactants. Step 1: synthesis of ethylene glycol-bis(2-chloroethano-
ate) (acylation). Step 2: synthesis of ethylene glycol bisacetyl-a,x-
dimethylalkylammonium chloride (quaternization).
n = 2, A = –C12 25, m = 12), G12-A3A-12 (n = 3, A = –C12
m = 12), G12-A4A-12(n = 4, A = –C H , m = 12), G14-A2A-14
G
12-A2A-12
Dodecyldimethyl tertiary amine, tetradecyldimethyl tertiary
amine, or hexadecyldimethyl tertiary amine (0.36 mol) and
acetone (50 g) were added into a 250-mL flask equipped with
a reflux condenser fixed on a magnetic stirrer. The solution
mixture was stirred and then the glycol-bis(2-chloroethano-
ate) (0.15 mol) was added to the flask. After refluxing for
(
H
25
H ,
12
25
33
(n = 2, A = –C14H29, m = 14), G16-A2A-16 (n = 2, A = –C16H ,
m = 16)
The peak assignments for the glycol-bis(2-chloroethanoate)
were as follows:
24 h, the mixture was placed at 4 °C until the product pre-
cipitated. The precipitate was washed several times with
-1
a) Diethylene glycol: O–H stretching (3,391 cm );
(
petroleum ether. It was dried in a vacuum oven at 45 °C for
C–H symmetric and asymmetric stretching
-1 -1
2,874 cm ); C–H bending (1,355–1,460 cm );
1
2 h to remove any trapped solvents to give the final product.
The yellow viscous liquids [G12-A2A-12, 96.2 % yield based
on diethylene glycol-bis(2-chloroethanoate); G12-A3A-12
5.1 % yield based on triethylene glycol-bis-(2-chloroet-
(
-1
H C–C–O–C (1,064, 1,120, 1,248 cm ).
2
,
(
b) Diethylene glycol-bis(2-chloroethanoate): O–H
-1
stretching (3,491 cm ); C–H symmetric and asym-
9
hanoate); G12-A4A-12, 93.5 % yield based on tetraethylene
glycol-bis(2-chloroethanoate); G14-A2A-14, 92.9 % yield
based on diethylene glycol-bis(2-chloroethanoate); and G16-
A2A-16, 91.3 % yield based on diethylene glycol-bis(2-chlo-
roethanoate)] were obtained [24, 25] (Scheme 1).
-1
metric stretching (2,860, 2,960 cm ); C=O stretch-
-1
(1,755 cm );
ing
C–H
bending
(1,354–
-1
,456 cm ); O=C–O–C symmetric and antisym-
1
-1
metric stretching (1,170–1,317 cm ), H C–C–O–C
2
-1
-1
(
1,034, 1,132 cm ); C–Cl stretching (700 cm ).
c) G12-A2A-12: C–H symmetric and asymmetric stretch-
(
Characterization
-1
ing
1,750 cm ); C–H bending (1,350–1,450 cm );
O=C–O–C symmetric and antisymmetric stretching
(2,850–2,920 cm );
C=O
stretching
-
1
-1
(
FTIR spectra were obtained on a Perkin-Elmer Spectrum
1
One. H NMR spectra were recorded on a Varian INOVA
-1
(
1,180–1,300 cm ),
H C–C–O–C
2
(1,035,
6
00 MHz NMR spectrometer with CDCl as the solvent
3
-1 -1
,137 cm ); O–H stretching (3,400 cm ) probably
1
and tetramethylsilane (TMS) as the internal reference. The
critical micelle concentration (CMC) was determined using
a DDSJ-308A conductivity meter.
arising from water because these surfactants were
very difficult to dehydrate.
Appearance of the C–Cl stretching band in the FTIR
FTIR of Diethylene Glycol-bis(2-Chloroethanoate)
and G12-A2A-12
spectra (b) compared with (a) confirmed the successful
synthesis of the intermediate product. Disappearance of
this band in the spectra for (c) confirmed that the syntheses
of these products was likely successful. Other FTIR spectra
FTIR spectroscopy was used to follow the synthetic reactions
of diethylene glycol-bis(2-chloroethanoate) and G12-A2A-12
.
of gemini surfactants were similar to those of G12-A2A-12.
1
23

J Surfact Deterg
a
3
b
2
c
2
f
2
d
2
?
N (CH
e
3
f
2
c
2
Further confirmations of successful syntheses were
1
obtained by H NMR spectroscopy.
assigned to [CH
h
(CH
g
)
11CH
CH
)
2
d
CH
g
h
?
N (CH
e
COOCH
b
CH
a
OCH
-
CH OCOCH
2
)
CH CH
2
2
2
2
3
2
(
CH ) CH ]Á2Cl .
2
11
3
1
H NMR of Diethylene Glycol-bis(2-Chloroethanoate),
(e) Diethylene glycol bisacetyl-a,x-bis (dimethylhexa-
decylammonium chloride) (G16-A2A-16): H NMR
1
Triethylene Glycol-bis(2-Chlorotetraethanoate),
and Ethylene Glycol-bis(2-Chloroethanoate)
(CDCl , 400 ppm), 0.88 (t, 6H, a), 1.25–1.76 (m,
2H, b), 2.84 (m, 4H, c), 2.95 (t, 4H, d), 3.54 (m,
3
5
1
a) Diethylene glycol-bis(2-chloroethanoate): H NMR
(
12H, e), 5.31 (s, 4H, f), 4.34 (t, 4H, g), 3.75 (t, 4H, h)
a
3
h
2
b
2
g
c
2
d
2
?
N (CH
e
3
(
CDCl , ppm), 4.12 (s, 4H, a), 4.35 (t, 4H, b),
3
were assigned to [CH
(CH
)
13CH
CH
f
)
2
a
f
g
2
b
2
h
2
?
N (CH
e
3
3
.74 ppm (t, 4H, c) were assigned to ClCH
a
CH
CH
2
d
2
COOCH
c
CH
OCH
a
CH
-
].2Cl .
2
OOCCH
2
)
2
2
b
c
c
b
COOCH CH OCH CH OOCCH Cl.
2
CH
2
(CH
)
13CH
3
2
2
2
2
1
(
b) Triethylene glycol-bis(2-chloroethanoate): H NMR
CDCl , ppm), 4.11 (s, 4H, a), 4.35 (t, 4H, b), 3.74 (t,
(
3
Antimicrobial Test
a
2
4
H, c), 3.66 (t, 4H, d) were assigned to ClCH
b
c
d
d
c
b
a
COOCH CH OCH CH OCH CH OOCCH Cl.
2
2
2
2
2
2
2
S. aureus and E. coli were lifted off with a wire loop and
1
(
c) Tetraethylene glycol-bis(2-chloroethanoate):
H
placed in 5 mL of TSB and incubated with shaking at
NMR (CDCl , ppm), 4.06 (s, 4H, a), 4.30 (t, 4H,
3
3
7 °C until the cultures became turbid. The turbid cultures
were then streaked out on TSA plates and incubated at
b), 3.69 (t, 4H, c), 3.61 (m, 8H, d) were assigned to
c
a
2
b
2
c
2
d
2
d
2
d
2
d
2
ClCH COOCH CH OCH CH OOCH CH OCH
2
3
7 °C for 24 h. A representative colony was lifted off with
a wire loop and placed in 5 mL of TSB, which was then
b
a
CH OOCCH Cl.
2
2
incubated with shaking at 37 °C for 24 h. At this stage, the
cultures of S. aureus and E. coli contained approximately
1
H NMR of Gemini Surfactants
9
0 CFU/mL and were used for antibacterial tests.
1
(
a) Diethylene glycol bisacetyl-a,x-bis (dimethyldodecy-
lammonium chloride) (G12-A2A-12): H NMR (CDCl ,
1
3
MIC and MBC Testing
ppm), 0.89 (t, 6H, a), 1.25–1.35 (m, 36H, b), 1.76 (m,
4
4
H, c), 3.80 (t, 4H, d), 3.56 (m, 12H, e), 5.32 (s, 4H, f),
a
The MIC and MBC tests against S. aureus and E. coli were
.34 (t, 4H, g), 3.75 (t, 4H, h) were assigned to [CH
b
3
performed using different concentrations of gemini sur-
-1
c
2
d
2
?
e
3 2
f
2
g
2
h
2
h
(
CH ) CH CH N (CH ) CH COOCH CH OCH
2
2
9
factants. A range of concentrations (2–1,024 lg mL ) for
each surfactant were prepared with 0.9 % sterilized, saline
g
2
f
2
?
e
3 2
d
2
c
2
b
2 9
a
3
-
CH OOCCH N (CH ) CH CH (CH ) CH ]Á2Cl .
(
b) Triethylene glycol bisacetyl-a,x-bis (dimethyldode-
cylammonium chloride) (G12-A3A-12): H NMR
5
water. The tested organisms (5 9 10 CFU, 50 lL of TSB)
1
were added to a 96-well microtiter plate. Each well con-
tained 350 lL of surfactant, 50 lL of TSB, and the test
organism. The 96-well plate was incubated at 37 °C for
(CDCl , ppm), 0.89 (t, 6H, a), 1.25–1.35 (m, 36H,
3
b), 1.76 (m, 4H, c), 3.65 (t, 4H, d), 3.62 (m, 12H, e),
5
.28 (s, 4H, f), 4.37 (t, 4H, g), 3.80 (t, 4H, h), 3.72
24 h. The 96-well plates were evaluated for growth of
a
t, 4H, i) were assigned to [CH (CH ) CH
3 2 9 2
b
c
(
bacterial cells and the lowest concentration of the anti-
bacterial agent where no visible growth of bacteria was
observed was determined to be the MIC. A small amount
of the mixture from each well was removed and spread on
solid agar plates. The plates were incubated at 37 °C for
d
2
?
e
3 2
f
2
g
2
h
2
i
2
i
h
CH N (CH ) CH COOCH CH OCH CH OCH
2
2
g
2
f
2
?
e
3 2
d
2
c
2
b
2 9
a
3
-
CH OOCCH N (CH ) CH CH (CH ) CH ]Á2Cl .
(
c) Tetraethylene glycol bisacetyl-a,x-bis (dimethyl-
dodecylammonium chloride) (G12-A4A-12): H NMR
1
(CDCl , ppm), 0.88 (t, 6H, a), 1.25–1.35 (m, 36H, b),
3
24 h to obtain MBC values. The MIC and MBC tests
1
5
.75 (m, 4H, c), 3.76 (t, 4H, d), 3.58 (m, 12H, e),
.20 (s, 4H, f), 4.40 (t, 4H, g), 3.80 (t, 4H, h), 3.67
were repeated at least four times for each antibacterial
surfactant. A aliquots of 400 lL of saline water and saline
water/TSB containing each test bacterium mixture (350/
a
b
c
d
2
i
(
m, 8H, i) were assigned to [CH (CH ) CH CH
?
3 2 9 2
i
e
3
f
2
g
2
h
2
i
2
i
N (CH ) CH COOCH CH OCH CH OCH CH
2
2
2
2
50 lL) were used as negative and positive controls,
h
2
g
2
f
2
?
e
3 2
d
2
c
2
b
2 9
a
3
OCH CH OOCCH N (CH ) CH CH (CH ) CH ]Á
respectively [26].
-
Cl .
2
(
d) Diethylene glycol bisacetyl-a,x-bis (dimethyltetra-
decylammonium chloride) (G14-A2A-14): H NMR
1
Time–Kill Study
(CDCl , ppm), 0.89 (t, 6H, a), 1.25–1.77 (m, 44H, b),
3
2
.85 (m, 4H, c), 3.13 (t, 4H, d), 3.56(m, 12H, e), 5.39
The time–kill study was performed to investigate the kill-
(s, 4H, f), 4.33 (t, 4H, g), 3.75 (t, 4H, h) were
ing kinetics of the antibacterial surfactants against S.
1
23

J Surfact Deterg
aureus and E. coli. A total of 4.5 mL of six continuous
mass concentrations of each antibacterial agent solution
were mixed with 0.5 mL of 0.9 % sterilized saline water
6
containing the bacterial suspension (5 9 10 CFU) in
culture tubes and used as the blank control. The tubes were
incubated at 37 °C for 24 h with shaking. After 24 h, a
small amount of solution was removed and the surviving
bacteria were then counted by dilution and spread-plate
methods. An aliquot of 45 mL of an accurate concentration
of the antibacterial agent solution was combined with 5 mL
of the 0.9 % sterilized saline water containing the bacterial
6
suspension (5 9 10 CFU) in a conical flask. The mixed
solution was incubated with shaking at 37 °C. After 10, 30,
6
0, 90, 120, 150, and 240 min of incubation, the surviving
bacteria were counted. These tests were repeated at least
three times [27].
Inhibitory Zone Method
A total of 15 mL of melted TSA was poured into a plate.
6
Then, 300 lL of cell suspension containing 2 9 10 CFU
was evenly spread on the surface of the agar plate. Round
filter paper (D = 6 mm) was placed on the agar plate and
then surfactants were added to the filter paper. Every plate
included a blank control. All plates were incubated at
3
7 °C for 3 days and the inhibition zone diameters were
then measured with vernier calipers. The inhibitory zone
test was repeated at least five times for each antibacterial
surfactant [28].
Fig. 1 CMC values of the cationic gemini surfactants. CMCs were
determined from electrical conductivity data (25 °C)
G12-A2A-12, G12-A3A-12, G12-A4A-12, G14-A2A-14, and G
16-A2A-16
-1
were 1.26, 1.85, 1.84, 1.80, and 1.38 mmol L , respec-
Results
tively. All five surfactants have low critical micelle concen-
trations [29]. It was observed that the CMC values did not
vary much with an increase in the length of the ethylene
glycol bisacetyl spacer and hydrophobic alkyl chain.
CMC Testing
The plot for conductance of these five gemini surfactants
in water is shown in Fig. 1. The conductivities of a range
of concentrations for each surfactant solution were found
to vary linearly with the surfactant concentration in both
premicellar and postmicellar regions. The ratio of the
slopes of the postmicellar to the premicellar region pro-
Antibacterial Tests
MIC and MBC Testing
vided the counterion dissociation constant (a ) and 1 -
cb
acb gives the counterion binding coefficient b . The
As shown in Table 1 (left panel), the MIC was the lowest
concentration with no visible growth. An aliquot from each
well was removed, spread on solid agar plates, and incu-
bated at 37 °C for 24 h to determine the MBC values
(Table 1, middle panel). Interestingly, the MIC values
obtained for E. coli or S. aureus were identical to the
obtained MBC values. Overall, the MIC and MBC results
showed that the antibacterial activities of the surfactants
against S. aureus were higher than those against E. coli.
The MIC and MBC values for G12-A2A-12, G12-A3A-12, and
cb
concentration at the intersection of these two lines was the
CMC of the corresponding surfactant [29]. The degree of
binding was related to the surface area per head group in
the ionic micelles. A lower value of the counterion
binding coefficient means a higher head group area [30]. bcb
values of G12-A2A-12, G12-A3A-12, G12-A4A-12, G14-A2A-14, and
G16-A2A-16 were 0.723, 0.698, 0.656, 0.706, and 0.651,
respectively. These results indicated that increasing the
length of the ethylene glycol bisacetyl spacer and hydro-
phobic alkyl chain caused a decrease of b . CMCs of
cb
G12-A4A-12 were not dependent on an increase in the glycol
1
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J Surfact Deterg
Table 1 MIC values, MBC
values, and inhibitory zone
diameters of five surfactants
against S. aureus and E. coli
-1
-1
Sample
MIC (lg mL
)
MBC (lg mL
)
Inhibitory zone diameter (mm)
S. aureus
E. coli
S. aureus
E. coli
S. aureus
E. coli
a
Blank
–
–
–
–
7.00 ± 0.02
24.01 ± 0.68
20.05 ± 0.44
21.2 8 ± 0.38
23.18 ± 0.75
14.82 ± 0.20
7.00 ± 0.02
12.33 ± 0.70
11.35 ± 0.52
10.83 ± 0.26
12.18 ± 0.56
7.08 ± 0.14
G
12-A2A-12
G
12-A3A-12
G
12-A4A-12
G
14-A2A-14
G
16-A2A-16
64
64
64
64
4
128
128
128
128
512
64
64
64
64
4
128
128
128
128
512
a
0
.9 % Saline water
Fig. 2 Plots of log (survivors) vs. concentration of G12-A2A-12
,
Fig. 3 Plots of log (survivors) vs. concentration of G12-A2A-12
,
G
12-A3A-12, and G14-A2A-14 against S. aureus after 24 h. Solid symbols
G
12-A3A-12, and G14-A2A-14 against E. coli after 24 h. Solid symbols
6
and solid lines cell suspension contained 10 cells/mL; hollow
6
and solid lines cell suspension contained 10 cells/mL; hollow
5
symbols and dotted lines cell suspension contained 10 cells/mL
5
symbols and dotted lines cell suspension contained 10 cells/mL
bisacetyl spacer chain length. Conversely, the MIC and
MBC values for G16-A2A-16 against S. aureus were signif-
icantly lower than those for G12-A2A-12. However, the val-
ues for G16-A2A-16 against E. coli were higher than for
G12-A2A-12 (see Supplementary Figs. 1 and 2).
parallel, the differences in the net increase in surfactant
5
concentrations toward both 10 CFU and 10 CFU of
6
E. coli (Fig. 3) suggested an antibacterial activity order of
G
12-A2A-12-[1 G14-A2A-14 [ G12-A3A-12
.
A
G
total
of
-1
64 lg mL
G
12-A2A-12, 96 lg mL
12-A3A-12, and
-
1
64 lg mL
G14-A2A-14 gave 100 % lethality toward
-1
5
Time–Kill Study
10 CFU of E. coli, whereas 80 lg mL
G
,
12-A2A-12
-
1
-1
9
6 lg mL G12-A3A-12, and 96 lg mL
G
were
14-A2A-14
6
required to achieve the same lethality against 10 CFU.
To further compare the antibacterial activity of the sur-
factants, the effects of various surfactant concentrations of
G12-A2A-12, G12-A3A-12, and G14-A2A-14 on the survival of S.
aureus were determined. As shown in Fig. 2, 100 %
The plots of surviving S. aureus cells vs contact time for
1
-
10 lg mL
of G12-A2A-12, G14-A2A-14, and G16-A2A-16
showed that it required only 10 min for G16-A2A-16 to kill
all bacteria, whereas the percentage survival of S. aureus in
the presence of G12-A2A-12 or G14-A2A-14 was still consid-
erable, even when exposed for 4 h (Fig. 4). However, the
sterilizing rate of G14-A2A-14 (about 80 %) was higher than
that of G12-A2A-12 (about 60 %). This result indicated that
the antibacterial activity of G16-A2A-16 against S. aureus
was significantly higher than that of either G12-A2A-12
or G14-A2A-14. In addition, the bacteriolytic effect of
5
lethality toward 10 CFU of S. aureus was achieved after
-
1
-1
treatment with 32 lg mL of G12-A2A-12, 56 lg mL of
-
1
G12-A3A-12, and 32 lg mL
0 lg mL of G12-A2A-12, 64 lg mL of G12-A3A-12, and
of G14-A2A-14, whereas
-1
-
1
4
-
1
3
2 lg mL
of G14-A2A-14 were required for similar
6
lethality in the presence of 10 CFU. Therefore, the dif-
ferent net increases of surfactant concentrations required to
obtain lethality reflected the antibacterial activities of the
surfactants: G14-A2A-14 [ G12-A2A-12 [ G12-A3A-12
.
In
G14-A2A-14 was slightly better than that of G12-A2A-12. The
1
23

J Surfact Deterg
Inhibitory Zone Method
The suppressing effects of the surfactants toward both S.
aureus and E. coli were further illustrated by measuring the
inhibitory zone diameters of the bacterial cultures after
exposure to the surfactants. The results confirmed that S.
aureus was more sensitive to these antibacterial agents
compared with E. coli (Table 1, right panel), which was in
good agreement with the results from the MIC/MBC test-
ing. On the basis of the size of inhibitory zone diameters,
all surfactants exhibited antibacterial activities against S.
aureus in the order G12-A2A-12 [ G14-A2A-14 [ G12-A4A-12
G12-A3A-12 [ G16-A2A-16, whereas against E. coli they were
in the order G12-A2A-12 [ G14-A2A-14 [ G12-A3A-12
[
[
G12-A4A-12 [ G16-A2A-16. The surfactants could also be
grouped as G12-A2A-12/G14-A2A-14, G12-A3A-12/G12-A4A-12, and
G16-A2A-16, representing those with high sensitivity, medium
sensitivity, and low sensitivity to the tested bacteria, respec-
tively (see Supplementary Fig. 3).
Fig. 4 Plots of log (S. aureus survivors) vs. contact time in a
5
suspension containing 5 9 10 CFU cells/mL in contact with
-1
1
0 lg mL of G12-A2A-12, G14-A2A-12, and G16-A2A-16
Discussion
In this work, a series of cationic gemini surfactants con-
taining two dimethylalkylammonium chains (two –C H ,
1
2 25
–C H , or –C H ) linked by diethylene glycol bisacetyl,
14 29 16 33
triethylene glycol bisacetyl, or tetraethylene glycol bis-
acetyl spacers were systematically synthesized by two
reactions, acylation and quaternization. Unlike previous
approaches, the synthetic methods discussed herein
allowed the study of different lengths of hydrophobic
chains and spacers [24, 25]. Acylation occurred via chlo-
roacetyl chloride instead of bromoacetyl bromide [16],
which produces environmentally harmful HBr. The yield of
the intermediate product ethylene glycol-bis(2-chloroet-
hanoate) was decreased by using chloroacetyl chloride;
however, the inductive effect of the oxygen atom of the
ether bond in the glycol does help to increase the yield.
Conversely, quaternization was a very safe and high
yielding reaction. Therefore, the advantages of this syn-
thetic strategy are safety, simplicity, and environmentally
friendly performance. The five products were obtained as
yellow viscous liquids at room temperature and improved
thermal stability (up to temperatures of 170 °C), similar to
ionic liquids [25].
Fig. 5 Plots of log (E. coli survivors) vs contact time in a suspension
5
-1
containing 5 9 10 CFU cells/mL in contact with 150 lg mL of
G12-A2A-12, G14-A2A-12, and G16-A2A-16
-
1
plots of surviving E. coli vs contact time for 150 lg mL
of G12-A2A-12, G14-A2A-14, and G16-A2A-16 showed that after
0 min, virtually all bacteria were killed by G12-A2A-12 and
1
G14-A2A-14 (Fig. 5). However, G16-A2A-16 showed limited
antibacterial activity (about 60 %) against E. coli with 4 h
of contact time at the same concentration, suggesting that
the antibacterial activity of G12-A2A-12 and G14-A2A-14
against E. coli was much higher than that of G16-A2A-16
under similar conditions. Therefore, an increase in the alkyl
chain length conferred a corresponding increase in the
antibacterial activity against S. aureus but a decrease in the
activity against E. coli.
The cell wall teichoic acid in S. aureus cells, the lipo-
polysaccharide (LPS) in E. coli cells, as well as the phos-
pholipids of both cells are rich in surface-exposed negative
charges. These negative charges allow the bis-quaternary
ammonium salt cationic gemini surfactants linked by a
ethylene glycol bisacetyl spacer to associate with the outer
surface of the target bacterial cells via electrostatic inter-
actions. Furthermore, the hydrophobic group of the
1
23

J Surfact Deterg
cationic gemini surfactants provided compatibility with the
hydrophobic end of the phospholipid of the cytoplasmic
membrane and allowed the cationic gemini surfactants to
be inserted into the cell membrane, resulting in disruption
of cell membrane integrity and eventually leading to
leakage of the intracellular lysate and dissolution of the
cytoplasmic membrane. Consequently, these surfactants
behaved as efficient sterilizers [30, 31]. Interestingly, the
MBC tests yielded values similar to the MIC values for
both bacteria. The bacteria were killed completely in the
time–kill study after only a 10-min exposure, indicating
that neither E. coli nor S. aureus was resistant to the gemini
surfactants [32]. On the basis of the results, the antibacte-
rial activities of the five surfactants against S. aureus were
higher than those against E. coli. Our current hypothesis to
explain this result is that because E. coli has a more
complicated overall cell wall structure than that of S.
aureus owing to its additional outer membrane, it is more
protected from antibacterial agents. Additionally, the
phospholipids in the E. coli outer membrane may make it
more difficult for the antibacterial agents to diffuse through
the membrane. As a result, the antibacterial activities of
these cationic gemini surfactants against E. coli were lower
than against S. aureus [33]. The order of antibacterial activ-
failed to induce a change in the charge density of the hydro-
philic portion.
Cationic gemini surfactants act as emulsifier and anti-
bacterial agents when added to emulsions [34]; their low
CMC and MIC/MBC values show that low additive
amounts can produce the expected antibacterial emulsions.
-
1
G12-A2A-12 had the lowest CMC, 1.26 mmol L , and
-
1
exhibited the best MICs of 32 lg mL toward S. aureus
-
1
5
and 64 lg mL toward E. coli in the presence of 10 CFU
of bacteria. Therefore, the optimized cationic gemini sur-
factant G12-A2A-12 containing two dodecyl chains linked by
a diethylene glycol bisacetyl spacer may have potential
applications as a quick-acting and efficient antibacterial
agent and emulsifier.
Acknowledgments The authors gratefully acknowledge financial
support from the National Major Scientific Instrument and Equip-
ment Development Project (No. 2011YQ12003505), the Department
of Education of Hubei Province (No. Q200510003), the Hubei
Province Nature Science Fund (No. 2011CDB061), and The Morn-
ing Program of Wuhan City Youth Science and Technology (No.
2
005ABA199).
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